Hydraulic Water Redistribution by Silver Fir (Abies Alba Mill.) Occurring Under Severe Soil Drought

Article Hydraulic Water Redistribution by Silver Fir (Abies alba Mill.) Occurring under Severe Soil Drought

1,2, , 3, 1 1 Paul Töchterle † * , Fengli Yang †, Stephanie Rehschuh , Romy Rehschuh , Nadine K. Ruehr 1, Heinz Rennenberg 3 and Michael Dannenmann 1

1 Institute of Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology (KIT), Kreuzeckbahnstrasse 19, 82467 Garmisch-Partenkirchen, Germany; [email protected] (S.R.); [email protected] (R.R.); [email protected] (N.K.R.); [email protected] (M.D.) 2 Institute of Geology, University of Innsbruck, Innrain 52f, 6020 Innsbruck, Austria 3 Institut für Forstbotanik und Baumphysiologie, Freiburg University, Georges-Köhler Allee 53/54, 79085 Freiburg, Germany; [email protected] (F.Y.); [email protected] (H.R.) * Correspondence: [email protected] These authors contributed equally to this study. † Received: 20 December 2019; Accepted: 29 January 2020; Published: 31 January 2020

Abstract: Hydraulic redistribution (HR) of water from wet- to dry-soil zones is suggested as an important process in the resilience of forest ecosystems to drought stress in semiarid and tropical climates. Scenarios of future climate change predict an increase of severe drought conditions in temperate climate regions. This implies the need for adaptations of locally managed forest systems, such as European beech (Fagus sylvatica L.) monocultures, for instance, through the admixing of deep-rooting silver fir (Abies alba Mill.). We designed a stable-isotope-based split-root experiment under controlled conditions to test whether silver fir seedlings could perform HR and therefore reduce drought stress in neighboring beech seedlings. Our results showed that HR by silver fir does occur, but with a delayed onset of three weeks after isotopic labelling with 2H O(δ2H +6000%), 2 ≈ and at low rates. On average, 0.2% of added 2H excess could be recovered via HR. Fir roots released water under dry-soil conditions that caused some European beech seedlings to permanently wilt. On the basis of these results, we concluded that HR by silver fir does occur, but the potential for mitigating drought stress in beech is limited. Admixing silver fir into beech stands as a climate change adaptation strategy needs to be assessed in field studies with sufficient monitoring time.

Keywords: hydraulic redistribution; drought; silver ﬁr; European beech; mixed stand

1. Introduction Hydraulic redistribution (HR) is the passive flux of water between wet- and dry-soil zones through plant roots as conduits. It is driven by soil-water potential gradients between dry- and wet-soil layers, and between roots and soil matrix [1,2]. Typically, HR occurs during the night, when transpiration has ceased [3–5]. Water can be redistributed in the upward (i.e., hydraulic lift [2,6,7]), downward (i.e., hydraulic descent [8–11]), and lateral directions [12–15]. Field observations showed that HR plays an important role in terrestrial ecohydrological cycles. Plants can benefit from HR through enhanced photosynthesis and transpiration [16], alleviated soil-moisture loss during the dry season [17], and a prolonged growing season [18,19]. These immediate benefits of HR consequently enhance nutrient acquisition [20], increase nutrient mobility, and facilitate root-litter decomposition [21,22].

Forests 2020, 11, 162; doi:10.3390/f11020162 www.mdpi.com/journal/forests Forests 2020, 11, 162 2 of 12

HR has been documented in more than 100 species [23], including agricultural crops and grasses [24–26], and forest trees and shrubs [27–29]. Although HR has been observed in diverse climatic settings [16,23,30,31], it is most prevalent in arid and semiarid ecosystems, such as savannas [16,32,33], arid climates and semideserts [5,34–36], Mediterranean-type ecosystems [10,37–39], and tropical forests [40–42]. However, HR may become increasingly relevant in temperate ecosystems that are subject to intensified drying–wetting cycles due to extreme drought events in projected climate-change scenarios [27,43]. Several techniques can be used to identify HR under laboratory or field conditions. Reverse water flow in roots can be identified by sap-flow techniques like the heat-balance [29,44] or -ratio method [45,46]. However, quantification of reverse water flow in roots by sap-flow measurements can easily be disrupted by fluctuations in ambient temperature [46,47], or small-scale soil and geomorphic variability [10]. Furthermore, sap flow can only be measured in individual roots and upscaling to the root system is a significant source of error [48]. Contrastingly, measurements of soil-water potential near plant roots, accompanied by water stable isotope analyses, have been used to track water movement in soil [30,49,50]. In this context, stable isotopes, either at natural abundance or by using heavy isotope 18 2 enriched water (i.e., H2 O, H2O) as a tracer, are used as a novel technique to quantify water flow between dry- and wet-soil layers through plant roots and water uptake by adjacent plants [3,27,51]. In temperate forests, HR was only detected in a few tree species in the field, such as Norway spruce (Picea abies (L.) Karst.), Douglas fir (Pseudotsuga menziesii), ponderosa pine (Pinus Ponderosa), loblolly pine (Pinus taeda), and sessile oak (Quercus petraea)[51–55]. In an adult mixed oak/European beech forest, Zapater et al. [51] showed HR by oaks using an 18O-labelling approach but did not find any tracer material in European beech. However, both HR and the uptake of redistributed water by neighboring plants was detected in studies with seedlings of English oak, Norway spruce, and European beech under moderate drought in split-root systems in the greenhouse [27]. In Central Europe, European beech, being both an abundant natural tree species and a key species in forestry, was reported to be particularly vulnerable to drought [56,57]. As extreme drought events and intensified drying–wetting cycles are projected to become more prevalent [43,58,59], beech forests in Central Europe face consequences such as declining growth and drastic economic losses for forestry [60–62]. Admixing deep-rooting tree species could potentially increase the resilience of beech stands. In this context, silver fir was proposed due to its high productivity and presumably higher drought resistance [63,64]. Furthermore, recent studies indicate that water supply to European beech in mixed forest stands may be supported by the presence of silver-fir neighbors [65,66]. The aim of this study was to show if silver fir can perform HR under extreme drought conditions. For this purpose, we applied an improved split-root approach under controlled conditions, combined 2 with the H2O labelling of water and in situ stable isotope analysis of soil moisture. We hypothesized 2 that fir roots were able to allocate H2O from moist- to dry-soil zones by HR.

2. Materials and Methods

2.1. Mesocosm Setup This study was conducted using plant-soil mesocosms under controlled conditions in the scientiﬁc greenhouse at the KIT Campus Alpin in Garmisch-Partenkirchen, Germany. Temperature (T) and relative humidity (rH) were controlled and underwent daily cycles (T = 20.5 4 C, and rH = 58 ± ◦ ± 12 % on average). Ambient CO2 concentration showed diurnal ﬂuctuations between 380 and 450 ppm without long-term trends during the timespan of the experiment. Six mesocosms, each comprising 2 nested polyvinylchloride (PVC) compartments (dimensions of inner and outer compartment: length width height = 90 38 40 cm3 and 20 20 10 cm3, × × × × × × respectively) were set up as shown in Figures1 and2. Soil for the mesocosms was collected in autumn 2015 in the Black Forest close to Emmendingen (SW Germany). The material was taken from the Ah horizon of a Dystric Cambisol that originated from Triassic sandstone and showed a sandy loam texture Forests 2020, 11, 162 3 of 12

texture (see [67] for site and soil details). The soil material was mixed with perlite at a volume ratio of 1:1. Perlite is a highly porous mineral that improves soil drainage and aeration properties while retaining moisture. These properties helped with the homogenization of soil moisture and isotope equilibration. Two silver-fir seedlings (3.5 years of age) were planted in the outer compartment (hereafter referred to as the fir compartment), and a single European beech seedling (2 years of age) in the inner compartment (hereafter referred to as the beech compartment). Root length of the fir and beech Forests 2020, 11, 162 3 of 12 Forestsseedlings 2020, 11 was, 162, on average, 30 and 15 cm, respectively. A first-order coarse root with intact second5 of 12- and third order fine roots of each silver fir seedling was redirected to the beech compartment. The A Picarro L 2130-i cavity ringdown spectrometer was used to analyze the stable isotopic (seemesocosms [67] for sitewere and then soil wrapped details). with The plastic soil material film to avoid was mixedevaporation with perliteand consequent at a volume contamination ratio of 1:1. composition (δ2H) of the gas stream. On measurement days, gas streams exiting each of the 6 Perliteof the islab a highlyenvironment porous with mineral 2H2O that vapor improves. The fir soil-root drainage strand ands were aeration the only properties hydraulic while connection retaining mesocosms were continuously measured for at least 15 min. To minimize carryover artefacts, lines moisture.between Thesebeech propertiesand fir compartment helped withs. theAs homogenizationexperiment control, of soil 1 mesocosm moisture andwas isotope left unplanted equilibration. but were flushed for 20 min between each measurement, and the values for the first 5 min of each otherwise received identical treatment (Figure 1). measurement were ignored. Stable isotope data reported in the results section of this study refer to the average values of the remaining measurement time. Results of each day were scale-normalized to 2 in-house standards (δ2H = –235.0 ± 1.8 ‰ and 1.8 ± 0.9 ‰ VSMOW) that were calibrated against international reference materials (VSMOW2, SLAP2). HR was quantified in this study as a percentage of excess deuterium (d) added to the fir compartment by labelling, that was recovered in the beech compartment. In other words, 2H excess recovery (∆d) corresponds to the needed amount of 2H from labelled water to reach the measured isotopic composition of a defined volume of water. We used isotopic measurements to calculate a mixing ratio between labelled water (i.e., δ2H = +6000‰) and background moisture using the isotopic composition of the control mesocosm as a baseline to account for natural variability. This mixing ratio is used to determine the amount of excess 2H in beech compartments, relative to excess 2H in the label water based on the VWC (ϴ) of the beech compartment and the amount of added label water (Equation 2):

= × 100 = 2 2 𝑚𝑚𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏ℎ × 100, (2) �𝜀𝜀 𝐻𝐻𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏ℎ−𝜀𝜀 𝐻𝐻𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐� 𝛳𝛳 ∆𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏ℎ 𝑚𝑚𝑂𝑂 2 2 𝑚𝑚𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 ∆𝑑𝑑𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 �𝜀𝜀 𝐻𝐻𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙−𝜀𝜀 𝐻𝐻𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐� 𝑀𝑀𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 ∆𝑑𝑑 𝑚𝑚𝑂𝑂 where ∆d is the percentage of 2H excess recovered in beech compartments, ∆dbeech and ∆dlabel are the 2 H excessFigureFigure [g] 1. 1. inExperiment Experiment the respective mesocosms.mesocosms. water Two reservoir fir fir and (i.e. 1 beech, beech saplingssapling compartments were planted or deuteratedin in polyvinylchloride polyvinylchloride label water), 2 2 2 ε Hbeech(PVC)(,PVC ε H) compartments, beechcompartments,, and ε Hbeech whereas whereas are the a beecha isotopic beech sapling sapling enrichment was was isolated isolated of within the within respective a smaller a smaller compartment water compartment reservoir in the in center.( giventhe in atomicAcente root % ofr strand. A deuterium root of strand each fir calculated of was each redirected fir fromwas intoredirected δ2H the values beech into),compartment. the beech and compartment. Soil moisture are the Soil was molar moisture extracted weight was via ratio 𝑚𝑚𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏ℎ 𝑚𝑚𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 diextractedffusion into via drydiffusion air passing into dry through air passing gas-permeable through tubinggas-permeable connected tubing to a waterconnected isotope to a analyser water between hydrogen and oxygen in the respective water𝑚𝑚𝑂𝑂 reservoir𝑚𝑚𝑂𝑂 at the measured isotopic composition,(Picarroisotope L analyserϴ 2130-i). is the Experiment (PicarroVWC of L beech comprised2130- i).compartments Experiment 6 such mesocosms; comprised, and M 3label were6 issuch usedthe mesocosmsabsolute as replicates amount; 3 and were 3 asof used controlslabel as water added(seereplicates to mainthe fir text and compartmen for 3 as more controls detail).t ([seeg]. main text for more detail).

2.2. Environmental Parameters Volumetric soil moisture and temperature were measured using DECAGON EM50 loggers (Decagon Devices, Inc., Pullman, Washington, DC, USA). A soil-moisture sensor (Type GS1 and 5TM, Decagon Devices, Inc., Pullman, WA, USA) was vertically installed in each beech compartment at approximately 10 cm depth. Volumetric water content (VWC) was recorded every 2 hours. Reported soil-moisture data were calibrated against gravimetric measurements of the same soil material used in the HR experiment. We observed erratic readings from soil-moisture sensors at very low soil-water contents. This had implications on the calculation of soil-water potential, as is explained in the following section.

2.3. Soil-Water Potential Soil-water potential was calculated for the beech compartments with a widely used model for water retention in soils [68]:

Figure 2. Photo of two mesocosms with silver-fir and European beech saplings in separate Figure 2. Photo of two mesocosms with silver-fir and European beech saplings in separate compartments. compartments. Two silver-fir seedlings (3.5 years of age) were planted in the outer compartment (hereafter 3. Results and Discussion referred to as the fir compartment), and a single European beech seedling (2 years of age) in the inner

Forests 2020, 11, 162 4 of 12 compartment (hereafter referred to as the beech compartment). Root length of the fir and beech seedlings was, on average, 30 and 15 cm, respectively. A first-order coarse root with intact second- and third order fine roots of each silver fir seedling was redirected to the beech compartment. The mesocosms were then wrapped with plastic film to avoid evaporation and consequent contamination of the lab 2 environment with H2O vapor. The fir-root strands were the only hydraulic connection between beech and fir compartments. As experiment control, 1 mesocosm was left unplanted but otherwise received identical treatment (Figure1).

2.3. Soil-Water Potential Soil-water potential was calculated for the beech compartments with a widely used model for water retention in soils [68]: (θs θr) θ = θr + − m (1) (1 + (α Ψ )n) | | 1  1  n  m  1  (θ θr) −  where Ψ =  − 1 , and Ψ is the absolute value of soil-water potential (hPa/cm water α  (θs θr)  | | ×  − −  3 3 column) at a speciﬁc volumetric water content θ (cm cm− ). θs and θr were the saturated and residual water content of the soil, respectively. Parameters α (hPa), n, and m are shape parameters. For ﬁtting, θs and θr were determined from measurements, and α and n were estimated using the soil texture of the potting substrate (sandy loam texture) according to Hodnett et al. [69]. For calculation, we used gravimetrically calibrated volumetric soil moisture. We conducted predawn water potential (Ψpredawn) measurements on 2 October to validate model results. Ψpredawn was measured on beech branches using a Scholander pressure chamber [70] (Model 1000 Pressure Chamber Instrument, PMS Instrument Company, Albany, Oregon, USA). Plant Ψpredawn could be used as an estimate for soil Ψ on the basis of the assumption that plant Ψpredawn was in equilibrium with soil Ψ adjacent to roots. Water potential is expressed as pF values, which is the logarithm of the absolute values of Ψ (hPa). Due to potential biases inherent to VWC measurements at very dry soil conditions, pF calculation can be subject to large uncertainty leading to pF values above the physical limit of ~6.9.

2.4. Leaf–Gas Exchange Measurements Leaf–gas exchange measurements on beech seedlings were performed from August to October, and captured the drought response of beech. We measured light-saturated photosynthesis (Asat) and stomatal conductance (gs) using a portable leaf–gas exchange system (Li-6400, LI-COR Inc., Lincoln, NE, USA) equipped with a light source (Li-6400-02B LED, LI-COR Inc., Lincoln, NE, USA). One green leaf per beech tree was measured under predetermined saturated light conditions (PAR = 1200 µmol 2 1 m− s− ), an average leaf temperature of 27.7 ◦C, and average relative humidity of 54%. Forests 2020, 11, 162 5 of 12

2.5. Hydraulic-Redistribution Experiment Prior to the experiment, the VWC of beech compartments was maintained at roughly 15% by irrigation. Each mesocosm was then exposed to drought conditions by withholding irrigation for 25 days. On the night from 27 to 28 August, 2 L (i.e., 5.8 L m 2) of deuterated water (δ2H +6000%) − ≈ was injected into the bottom-soil zone of the fir compartment of 3 mesocosms containing trees and the unplanted control mesocosm. For each fir compartment, 6 evenly spread-out irrigation tubes (Figure1) were used as injection ports in order to ensure the homogeneous distribution of deuterated water. The two remaining mesocosms containing trees received no label injection. Isotopic composition of soil moisture in the beech compartment was measured on 15 separate days during the 10 following weeks. Measurements generally took place during the morning hours until noon. Each beech compartment was re-watered on 29 October with 1.5 L of tap water in order to weaken the water-potential gradient between the fir and beech compartments, and, thus, the required conditions for HR.

2.6. Deuterated-Water Tracing We used a membrane-inlet water-vapor sampling technique in soil coupled to a cavity ringdown 2 laser spectrometer [71,72] to trace H2O transport from the fir to the beech compartment. For this purpose, the beech compartment of each mesocosm was equipped with gas-permeable tubing (Accurel PP V8/2HF polypropylene tubing, Membrana GmbH, Germany: 30 cm long sections buried at 5 and 10 cm depth) that was flushed with synthetic dry air (20% O2 in N2) during measurements (Figure2). The dry air absorbed moisture upon passing the gas-permeable section. This approach resembled in situ soil air probes [72], but with double the length of gas-permeable tubes in each beech compartment. Accurel tubes are suitable for isotopic measurements, as they do not cause isotopic fractionation across a wide range of soil-moisture contents, and render possible vapor measurements in equilibrium with soil water [71]. A Picarro L 2130-i cavity ringdown spectrometer was used to analyze the stable isotopic composition (δ2H) of the gas stream. On measurement days, gas streams exiting each of the 6 mesocosms were continuously measured for at least 15 min. To minimize carryover artefacts, lines were flushed for 20 min between each measurement, and the values for the first 5 min of each measurement were ignored. Stable isotope data reported in the results section of this study refer to the average values of the remaining measurement time. Results of each day were scale-normalized to 2 in-house standards (δ2H = –235.0 1.8 % and 1.8 0.9 % VSMOW) that were calibrated against international ± ± reference materials (VSMOW2, SLAP2). HR was quantified in this study as a percentage of excess deuterium (d) added to the fir compartment by labelling, that was recovered in the beech compartment. In other words, 2H excess recovery (∆d) corresponds to the needed amount of 2H from labelled water to reach the measured isotopic composition of a defined volume of water. We used isotopic measurements to calculate a mixing ratio between labelled water (i.e., δ2H = +6000%) and background moisture using the isotopic composition of the control mesocosm as a baseline to account for natural variability. This mixing ratio is used to determine the amount of excess 2H in beech compartments, relative to excess 2H in the label water based on the VWC (θ) of the beech compartment and the amount of added label water (Equation 2): 2 2 mbeech ∆d ε Hbeech ε Hcontrol m θ ∆d = beech 100 = − O 100, (2) 2 2 mlabel ∆dlabel × (ε Hlabel ε Hcontrol) Mlabel × − mO 2 where ∆d is the percentage of H excess recovered in beech compartments, ∆dbeech and ∆dlabel are the 2H excess [g] in the respective water reservoir (i.e., beech compartment or deuterated label water), 2 2 2 ε Hbeech, ε Hbeech, and ε Hbeech are the isotopic enrichment of the respective water reservoir (given in atomic % of deuterium calculated from δ2H values), mbeech and mlabel are the molar weight ratio between mO mO hydrogen and oxygen in the respective water reservoir at the measured isotopic composition, θ is Forests 2020, 11, 162 6 of 12

the VWC of beech compartments, and Mlabel is the absolute amount of label water added to the ﬁr compartment [g].

3. Results and Discussion Forests 2020, 11, 162 6 of 12 After 25 days of drought treatment, the VWC in the planted beech compartments declined to 6% 2%,After while 25 days VWC of drought in the unplanted treatment, control the VWC compartment in the planted was beech still at compartments 16%. Soil-water declined potential to 6 in% ± the± 2%, beech while compartments VWC in the ofunplanted the mesocosms control rangedcompartment between was pF still values at 16%. of ca. Soil 2.0-water and 3.5potential (Figure in3 c).the beech compartments of the mesocosms ranged between pF values of2 ca.1 2.0 and 3.5 (Figure 3c). Net Net beech photosynthesis had declined to an average of 8 µmol m− s− (Figure3a), and stomatal beech photosynthesis had declined to an average2 1of 8 µmol m-2 s-1 (Figure 3a), and stomatal conductance showed values of around 0.14 mol m− s− (Figure3b). After the ﬁr compartments were labelledconductance with 2showed L of deuterated values of around water, the0.14 net mol photosynthesis m-2 s-1 (Figure and3b). After stomatal the conductancefir compartments of beech were labelled with 2 L of deuterated water,2 1 the net photosynthesis2 1 and stomatal conductance of beech continued to decline to 4 µmol m− s− and 0.05 mol m− s− , respectively, one week after labelling. continued to decline to 4 µmol m-2 s-1 and 0.05 mol m-2 s-1, respectively, one week after labelling.

Figure 3. (a) Time series of light-saturated photosynthesis (Asat) and (b) stomatal conductance (gs) Figure 3. (a) Time series of light-saturated photosynthesis (Asat) and (b) stomatal conductance (gs) of of beech seedlings as measurement averages standard error (n = 5). (c) Soil matrix potential in beech seedlings as measurement averages ± standard± error (n = 5). (c) Soil matrix potential in beech beech compartments (daily means) as pF-values, log(|hPa|) for three labelled (black, grey, light grey), compartments (daily means) as pF-values, log(|hPa|) for three labelled (black, grey, light grey), two two unlabelled (green, light green), and one unplanted control (light blue) mesocosms. Solid vertical unlabelled (green, light green), and one unplanted control (light blue) mesocosms. Solid vertical line: line: time of deuterium labelling; dotted vertical line: re-watering of beech compartments. Very high time of deuterium labelling; dotted vertical line: re-watering of beech compartments. Very high pF pF values of some replicates from 10 April onwards were likely related to volumetric-water-content values of some replicates from 10 April onwards were likely related to volumetric-water-content (VWC) measurement bias in very dry soil conditions. (VWC) measurement bias in very dry soil conditions. These data showed that the beech trees experienced severe drought stress during the experiment These data showed that the beech trees experienced severe drought stress during the experiment that was not significantly counteracted by the silver-fir seedlings. Previous studies also reported a that was not significantly counteracted by the silver-fir seedlings. Previous studies also reported a similar development for mature trees in the field under severe drought conditions at mixed fir and similar development for mature trees in the field under severe drought conditions at mixed fir and beech cultivation [66]. beech cultivation [66]. In unlabeled mesocosms and the labelled control mesocosm, δ2H values of soil moisture in the In unlabeled mesocosms and the labelled control mesocosm, δ2H values of soil moisture in the beech compartment remained between –120% and –80% VSMOW throughout the entire incubation beech compartment remained between –120‰ and –80‰ VSMOW throughout the entire incubation period (Figure4a). Labelled mesocosms showed similar or slightly enriched values for the first three period (Figure 4a). Labelled mesocosms showed similar or slightly enriched values for the first three weeks after labelling. In addition, 2H excess recovery in the beech compartments was negligible during weeks after labelling. In addition, 2H excess recovery in the beech compartments was negligible that time. This suggested that no detectable HR occurred for three weeks after label injection. during that time. This suggested that no detectable HR occurred for three weeks after label injection.

ForestsForests 20202020,, 1111,, 162 162 7 7of of 12 12

Figure 4. (a) Time series of δ2H in soil moisture in beech compartment of 2H-labeled mesocosms (grey), Figure 4. (a) Time series of δ2H in soil moisture in beech compartment of 2H-labeled mesocosms planted unlabelled mesocosms (green), and labelled control mesocosm without plants (light blue). (grey), planted unlabelled mesocosms (green), and labelled control mesocosm without plants (light Dashed lines: time series of each respective replicate; solid lines: their mean values. Re-watering blue). Dashed lines: time series of each respective replicate; solid lines: their mean values. Re-watering was with unlabelled water. (b) Time series of 2H excess recovery in beech compartments expressed was with unlabelled water. (b) Time series of 2H excess recovery in beech compartments expressed as as percentage of 2H excess added by deuterated water to fir compartment. Isotopic composition of percentage of 2H excess added by deuterated water to fir compartment. Isotopic composition of control mesocosms’ beech compartment used as baseline for excess calculation. Grid was spaced at control mesocosms’ beech compartment used as baseline for excess calculation. Grid was spaced at 14 day intervals. 14 day intervals. During this initial three-week period, soil pF further increased in the beech compartments During this initial three-week period, soil pF further increased in the beech compartments (Figure3c). Values for δ2H in the beech compartments started to notably rise afterwards (Figure4a). (Figure 3c). Values for δ2H in the beech compartments started to notably rise afterwards (Figure 4a). On the basis of 2H excess recovery, we could assume that traceable amounts of water were reallocated On the basis of 2H excess recovery, we could assume that traceable amounts of water were reallocated to the beech compartment through the fir roots (Figure4b). Hence, HR occurred in this experiment with to the beech compartment through the fir roots (Figure 4b). Hence, HR occurred in this experiment a delay of ca. 2–3 weeks after label application. However, this delayed HR occurred at pF values above with a delay of ca. 2–3 weeks after label application. However, this delayed HR occurred at pF values the permanent wilting point (i.e., pF = 4.2 according to Amelung et al. [73]; Figure3c), and partially above the permanent wilting point (i.e., pF = 4.2 according to Amelung et al. [73]; Figure 3c), and resulted in the wilting of the beech seedlings. Seedling wilting is also represented by the concurrent partially resulted in the wilting of the beech seedlings. Seedling wilting is also represented by the drop of photosynthetic activity and stomatal conductance to values close to zero (Figure3a,b). concurrent drop of photosynthetic activity and stomatal conductance to values close to zero (Figure Prior to re-watering, 2H excess recovered in the beech compartments amounted to an average 3a,b). of 0.2% of 2H excess added with the 2 L of deuterated water (Figure4b). Re-watering of the beech Prior to re-watering, 2H excess recovered in the beech compartments amounted to an average of compartments with 1.5 L of water at the end of October clearly diluted the δ2H signal (Figure4a). 0.2% of 2H excess added with the 2 L of deuterated water (Figure 4b). Re-watering of the beech Furthermore, pF values increased above the permanent wilting point. However, 2H excess recovery compartments with 1.5 L of water at the end of October clearly diluted the δ2H signal (Figure 4a). was initially doubled by re-watering, followed by rapid decline (Figure4b). We assumed that the Furthermore, pF values increased above the permanent wilting point. However, 2H excess recovery unexpected doubling of 2H excess upon re-watering was caused by overestimated VWC measurements was initially doubled by re-watering, followed by rapid decline (Figure 4b). We assumed that the directly after re-watering the beech compartments (water amount was part of the calculation of 2H unexpected doubling of 2H excess upon re-watering was caused by overestimated VWC excess recovery; see Equation 2). As indicated by δ2H dynamics, re-watering seemed to eliminate measurements directly after re-watering the beech compartments (water amount was part of the the large gradient in water potential, which was needed to sustain HR through water loss from the calculation of 2H excess recovery; see Equation 2). As indicated by δ2H dynamics, re-watering seemed fir roots. to eliminate the large gradient in water potential, which was needed to sustain HR through water Overall, δ2H was correlated to pF values in the beech compartments (Figure5). This suggests loss from the fir roots. that water-potential gradients largely drove HR by the silver-fir roots, and HR was stronger at high Overall, δ2H was correlated to pF values in the beech compartments (Figure 5). This suggests pF values. A similar observation was made in a loblolly pine stand, where hydraulic redistribution that water-potential gradients largely drove HR by the silver-fir roots, and HR was stronger at high was determined by reverse root flow and was shown to increase with soil drought [74]. However, pF values. A similar observation was made in a loblolly pine stand, where hydraulic redistribution in split-root experiments for Norway spruce, European beech, and English oak, Hafner et al. [27] was determined by reverse root flow and was shown to increase with soil drought [74]. However, in observed hydraulic redistribution already under moderate drought (water potential of ca. –0.5 MPa/ split-root experiments for Norway spruce, European beech, and English oak, Hafner et al. [27] –5000 hPa/pF value of 3.7) after only several days. In these conditions, we could not find evidence observed hydraulic redistribution already under moderate drought (water potential of ca. –0.5 MPa/ to support HR at similar time scales. Still, our results were consistent with the observation that, in –5000 hPa/pF value of 3.7) after only several days. In these conditions, we could not find evidence to support HR at similar time scales. Still, our results were consistent with the observation that, in a

Forests 2020, 11, 162 8 of 12

Forests 2020, 11, 162 8 of 12 a mature mixed beech–fir forest, the presence of fir slightly improved the xylem-sap flow density ofmature beech mixed compared beech to–fir a beechforest, monoculturethe presence of [66 fir]. However,slightly impr thisoved improvement the xylem-sap was flow not enoughdensity toof counteractbeech compared the negative to a beech effect ofmonoculture drought on xylem-sap[66]. However, flow density.this improvement In addition, was thepositive not enough effect ofto thecounteract presence the of negative fir on xylem-sap effect of drought flow density on xylem in beech-sap uponflow density. drought In was addition, reversed the when positive the foresteffect standof the waspresence subjected of fir toon precipitationxylem-sap flow after density drought. in beech These upon data drought indicated was that reversed cocultivation when the of beechforest andstand fir was may subjected not enhance to precipitation the resilience after of beech drought. to drought These eventsdata indicate throughd that HR. cocultivation of beech and fir may not enhance the resilience of beech to drought events through HR.

FigureFigure 5.5. Relationship between δ2HH and and pF value valuess in beech compartments of labelled mesocosms. Only incubation period from labelling untiluntil re-wateringre-watering waswas considered.considered.

4.4. Conclusions 2 We setset up a split-rootsplit-root experiment with silver-firsilver-fir andand EuropeanEuropean beechbeech seedlingsseedlings usingusing 2H labelled water to tracktrack HRHR fromfrom wet-wet- toto dry-soildry-soil zoneszones throughthrough silver-firsilver-fir roots.roots. In situ measurements of the isotopic composition of of soil soil moisture moisture show showeded detectable detectable HR HR at at2– 2–33 weeks weeks after after label label application. application. A Apositive positive correlation correlation between between soil soil pF pF values values and and the the isotopic isotopic composition composition of of soil soil moisture moisture confirm confirmeded 2 that changes in waterwater potentialpotential drivedrive HR.HR. ConsideringConsidering that only 0.2%0.2% ofof thethe addedadded 2H could be recovered at the permanentpermanent wilting point, we concludedconcluded that the admixing of silver firfir toto EuropeanEuropean beech standsstands likely likely does does not not increase increase the resiliencethe resilience of beech of monoculturesbeech monocultures to severe droughtto severe conditions. drought Theconditions. delayed The onset delayed of HR onset of 2–3 of weeks HR of in 2 this–3 weeks study in led this us tostudy recommend led us to thatrecommend future field that studies future allow field forstudies a longer allow monitoring for a longer time monitoring to fully detect time to hydraulic fully detect redistribution. hydraulic redistribution.

Author Contributions:Contributions:Conceptualization, All authors have H.R.,read F.Y.,and P.T., agree andd M.D.;to the methodology, published version P.T., F.Y., of H.R., the M.D.,manuscript. N.K.R.; dataConceptualization processing, P.T.,, H F.Y.,.R., S.R.,F.Y., R.R.;P.T., writing—original-draftand M.D.; methodology, preparation, P.T., F.Y., P.T.,H.R. F.Y.,, M.D M.D.,., N.K H.R.,.R.; data S.R.; processing writing—review, P.T., andF.Y., editing, S.R., R.R all.; authors;writing— visualization,original-draft P.T., preparation, S.R.; supervision, P.T., F.Y H.R.,., M.D M.D.,., H.R N.K.R.., S.R.; Allwriting authors—review have read and andediting, agreed all to the published version of the manuscript. authors; visualization, P.T., S.R.; supervision, H.R., M.D., N.K.R. All authors have read and agreed to the Funding:published versionThis study of the was manuscript. funded by the Federal Ministry of Food and Agriculture (BMEL) within the “Waldklimafonds” program and the “Buchen–Tannen–Mischwälder zur Anpassung von Wirtschaftswäldern an ExtremereignisseFunding: This study des Klimawandelswas funded by (BuTaKli)” the Federal project; Ministry their supportof Food is gratefullyand Agriculture acknowledged. (BMEL) Additionalwithin the funding‘‘Waldklimafonds’’ was provided program by the Germanand the Federal‘‘Buchen Ministry–Tannen of– EducationMischwälder and zur Research Anpassung (BMBF) von through Wirtschaftswäldern the Helmholtz Associationan Extremereignisse and its ATMO des Klimawandels research program. (BuTaKli)’’ RR andproject NKR; the acknowledgeir support is gratefully support from acknowledged. the German Additional Research Foundation through its Emmy Noether Program (RU 1657/2-1). funding was provided by the German Federal Ministry of Education and Research (BMBF) through the Acknowledgments:Helmholtz AssociationWe and would its likeATMO to thank research Andreas program Gast,. R NilsR and Risse, NKR and acknowledge Stefanie Dumberger support forfrom their the assistance German withResearch the experimentFoundation setupthrough and its leaf–gas Emmy exchangeNoether Program measurements. (RU 1657/2 We-1). would also like to acknowledge the two anonymous reviewers whose comments have greatly improved the manuscript. ConﬂictsAcknowledgments: of Interest: WeThe would authors like declare to thank no conﬂict Andreas of interest. Gast, Nils Risse, and Stefanie Dumberger for their assistance with the experiment setup and leaf–gas exchange measurements. We would also like to acknowledge the two anonymous reviewers whose comments have greatly improved the manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

Forests 2020, 11, 162 9 of 12

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